An essential step in viral replication is the assembly of the viral particle. For retroviruses, this step involves viral structural proteins, the viral genome, lipid membranes and possibly host factors. This proposal continues a long term interest of the laboratory, the assembly of avian sarcoma and leukemia virus. The focus of the research will be the Gag protein which provides the internal structural proteins. Four major directions will be followed. The first involves studying the assembly of virus-like particles in vitro. An in-vitro assembly system has been developed in the laboratory which yields hollow cylinders or spherical particles, depending on the sub-fragment of Gag utilized. The size and nature of the particles will be further characterized and the functional role of different domains of Gag defined. Additional studies will define the role of RNA in the assembly process. The second aim studies the regulation of proteolysis. The Gag and Gag-Pol proteins are cleaved by the viral protease. Studies will focus on the amino acid sequences N-terminal to the protease domain to determine if and how they regulate proteolysis and dimerization. The role of the cellular membrane in proteolysis will also be investigated. The third aim studies RNA packaging. The encapsidation signal of the RNA is contained in the 5' portion of the viral genome. Experiments are proposed in which short sequences within E (or psi) will be randomized. Sequences capable of functioning will be selected in vivo by packaging into viral particles as well as in vitro using purified proteins. The final aim involves defining the structure of viral proteins by X-ray crystallography and particles assembled in vitro by cryo-electron microscopy. Purified protein and assembled particles will be provided to collaborators who are experts in the techniques.